Prior to the present invention, cell-based assays for toxins such as Botulinum neurotoxins (BoNT) are limited to cells that contain the appropriate surface receptors used by the specific toxin to gain entry into cells. The efficiency of toxin entry into immortalized cell lines is often much poorer than that obtained with primary cultures, requiring the use of high concentrations of toxin and acceptance that a low percentage of the immortalized cells will become intoxicated. The use of holotoxin, the native toxin, in screening adds a substantial complication due to additional regulatory, safety and waste disposal issues.
Also, cell-based assays for toxins can be important in the development of therapeutic antitoxins and pharmacological antidotes, and in some aspects of diagnostic test development. Such assays are generally limited by several features. First, many cell lines are insensitive to a toxin, probably because they lack one or more surface receptors necessary for the toxin to enter the cell. Second, even when immortalized cell lines, such as neuroblastoma lines, are susceptible to intoxication, they are generally much less sensitive than primary neuronal cells. Thirdly, most cell lines that are sensitive to intoxication by one toxin are insensitive to most other toxins, limiting their broad utility. Finally, the use of holotoxin in the assays, that requires substantial safety and regulatory issues, severely complicate their use in high-throughput screening assays, e.g., it is difficult to get enough toxin to perform the assays.
Hence, a need exists for efficient ways to intoxicate one or more cells without having to use high concentrations of a holotoxin. A further need exists to intoxicate cells that are traditionally considered to be insensitive or refractory to toxins, and to intoxicate more than one cell type with the same toxin. Yet a further need exists to be able to intoxicate cells without using the entire toxin, but rather the enzymatically active portion thereof to avoid regulatory, safety and waste disposal issues.